The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
The art of attachment chemistry for macroscopic and microscopic arrays has received much attention in recent years. However, as requirements for assay sensitivities have increased, the need for attachment chemistries that are able to provide greater specificity in attachment of biomolecules, as well as greater density of attached biomolecules, has also increased.
Although covalent and non-covalent attachment chemistries have become well developed, few advances have overcome some of the fundamental difficulties experienced with electronic microarrays. For example, problems with present electronic microarrays include non-specific binding of biomolecules outside specific capture/detection sites and the inability to control passive binding of such biomolecules before, during, and after electronic addressing. These problems result in less than ideal discrimination of target molecules from nontarget molecules. Likewise, attachment chemistries currently in use have made it difficult to manipulate unused capture sites during multiple site or sequential site addressing of target molecules without encountering undesired passive binding of nontarget molecules to the capture sites.
In providing a solution to the problems of low specificity of binding and discernment of target from nontarget, we provide attachment chemistry for attaching biomolecules that provides specificity of binding as well as enhanced target discernment at the capture sites by a method which significantly increases the density of target binding sites on the microarray. Moreover, this method provides for localizing the deposition of target binding sites using the electrodes of the microarray.
While much of the art involving microarray deposition concerns masking techniques to enhance specificity of binding, one method has been used to electrochemically treat the surface desired to be patterned with chemical moieties. In PCT application WO93/22480 by Southern is disclosed a method of electrochemically patterning a surface wherein an electrode grid is positioned adjacent to the surface desired to be patterned in solution and in direct contact with the electrolyte. The electrode grid provides an electronic potential that directs deposition of molecules, removal of molecules, or chemical modification of molecules, on the substrate surface. Following such electronic treatment process, the grid is removed. Although such a method provides a means for patterning a nonelectronic surface, it is applicable only to passive array formats. Additionally, the method can only be applied in initial manufacture of an array and not applied to an array in an active “on-demand” manner.
Other art has centered on deposition chemistry. For example, Sundberg et al. in U.S. Pat. No. 5,919,523 discloses attachment chemistry typically used for attaching molecules directly to derivatized glass slides. Such chemistry is designed for attaching and synthesizing nucleic acid sequences wherein one useful aspect is a need for wettability of the substrate surface. Such chemistry is further distinguished from that used in the present invention in that the electronic microarrays of the present invention require a porous permeation layer above the electrodes which comprises reactive moieties that participate in the attachment and polymerization chemistry scheme of the invention.
In both of the above examples, as in the case with typical attachment chemistries, the levels of binding of attached molecules is limited to the number of reactive moieties present on the substrate surface. Thus, there remains a need in general for a method of derivatizing a substrate surface in such a way that attachment sites for binding molecules of interest to the substrate is dramatically increased. We have found such a method with respect specifically to substrates comprising permeation layers of electronically addressable microarrays.
As is well understood in the art of hybridization and detection of target molecules using nucleic acid probes, it is important to have a high density of capture probe binding on the detection surface of the microarray. In conventional surface modification techniques, the available groups on the surface are derivatized to include functional moieties that can bind to capture probes. The capture probe binding in turn depends on the number and availability of derivatized groups or binding sites on the detection surface. In many applications, such as infectious disease detection, genomic research, etc., the ability to detect very low levels of nucleic acid is necessary. To this end, the current invention provides a novel method of surface modification wherein polymers of functional groups having multiples of attachment moieties for binding capture probes (i.e. derivatized biomolecules) are on the surface of the permeation layer. The availability of such attachment moieties may also be placed at predetermined positions on the array. This provides for increased discrimination between specific and nonspecific binding of biomolecules on the microarray.